Scalable Microgravity Simulator Used for Long-Term Musculoskeletal Cells and Tissue Engineering

Since opportunities to conduct experiments in space are scarce, various microgravity simulators and analogs have been widely used in space biology ground studies. Even though microgravity simulators do not produce all of the biological effects observed in the true microgravity environment, they provide alternative test platforms that are effective, affordable, and readily available to facilitate microgravity research. The Microgravity Simulation Support Facility (MSSF) at the National Aeronautics and Space Administration (NASA) John F. Kennedy Space Center (KSC) has been established for conducting short duration experiments, typically less than 1 month, utilizing a variety of microgravity simulation devices for research at different gravity levels. The simulators include, but are not limited to, 2D Clinostats, 3D Clinostats, Random Positioning Machines, and Rotating Wall Vessels. In this chapter, we will discuss current MSSF capabilities, development concepts, and the physical characteristics of these microgravity simulators.

This study focuses on the effects of short-term [22 s, parabolic flight campaign (PFC)] and long-term (10 d, Shenzhou 8 space mission) real microgravity on changes in cytokine secretion and gene expression patterns in poorly differentiated thyroid cancer cells. FTC-133 cells were cultured in space and on a random positioning machine (RPM) for 10 d, to evaluate differences between real and simulated microgravity. Multianalyte profiling was used to evaluate 128 secreted cytokines. Microarray analysis revealed 63 significantly regulated transcripts after 22 s of microgravity during a PFC and 2881 after 10 d on the RPM or in space. Genes in several biological processes, including apoptosis (n=182), cytoskeleton (n=80), adhesion/extracellular matrix (n=98), proliferation (n=184), stress response (n=268), migration (n=63), angiogenesis (n=39), and signal transduction (n=429), were differentially expressed. Genes and proteins involved in the regulation of cancer cell proliferation and metastasis, such as IL6, IL8, IL15, OPN, VEGFA, VEGFD, FGF17, MMP2, MMP3, TIMP1, PRKAA, and PRKACA, were similarly regulated under RPM and spaceflight conditions. The resulting effect was mostly antiproliferative. Gene expression during the PFC was often regulated in the opposite direction. In summary, microgravity is an invaluable tool for exploring new targets in anticancer therapy and can be simulated in some aspects in ground-based facilities.

The main goal of this research is to further contribute to the validation of the clinostat Random Positioning Machine (RPM) for vivo experiments by studying bone marrow cells mineralization in the rat. These cells were obtained from the bone shafts of the femora and cultured for 13 days. The production of calcified matrix was analyzed by Alizarin Red staining demostrating that calcium deposition was significantly decreased in RPM samples. Moreover, we demostrated what Alkaline phosphatase (ALP) activity, a marker of osteoblastic phenotype, is down regulate by simulated microgravity. Also in vitro experiments in coltured osteoblasts (first subcolture) exposed to RPM for 9 days confirm a strongly decreased mineralization and differentiation.

During in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) hypertrophy is an inadvertent event associated with cell differentiation toward the osteogenic lineage. Up to now, there is no stringent experimental control mechanism to prevent hypertrophy of MSCs. Microgravity is known to have an impact on osteogenesis. In this study, the influence of simulated microgravity (SMG) on both chondrogenesis and hypertrophy of hMSCs was evaluated. A bioreactor using a rotating wall vessel was constructed to simulate microgravity. Pellet cultures formed from hMSCs (P5) were supplemented with human transforming growth factor-β3 (TGF-β3). The hMSC pellet cultures treated with TGF-β3 were either kept in SMG or in a control system. After three weeks of culture, the chondrogenic differentiation status and level of hypertrophy were examined by safranin-O staining, immunohistochemistry and quantitative real-time PCR. SMG reduced the staining for safranin-O and collagen type II. The expression of collagen type X α1 chain (COL10A1) and collagen type II α1 chain (COL2A1) were both significantly reduced. There was a higher decrease in COL2A1 than in COL10A1 expression, resulting in a low COL2A1/COL10A1 ratio. SMG reduced hypertrophy of hMSCs during chondrogenic differentiation. However, the expression of COL2A1 was likewise reduced. Even more, the COL2A1/COL10A1 ratio decreased under SMG conditions. We therefore assume that SMG has a significant impact on the chondrogenic differentiation of hMSCs. However, due to the high COL2A1 suppression under SMG, this culture system does not yet seem to be suitable for a potential application in cartilage repair.

Microgravity has been shown to induces many changes in proliferation, differentiation and growth behavior of stem cells. Little is known about the effect of microgravity on hematopoietic differentiation of pluripotent stem cells (PSCs). In this study, we used the random position machine (RPM) to investigate whether simulated microgravity (SMG) allows the induction of hematopoietic stem/progenitor cell (HSPC) derived from human embryonic stem cells (hESCs) in vitro. The results showed that SMG facilitates hESCs differentiate to HSPC with more efficient induction of CD34+CD31+ hemogenic endothelium progenitors (HEPs) on day 4 and CD34+CD43+ HSPC on day 7, and these cells shows an increased generation of functional hematopoietic cells in colony-forming unit assay when compared with normal gravity (NG) conditions. Additionally, we found that SMG significantly increased the total number of cells on day 4 and day 7 which formed more 3D cell clusters. Transcriptome analysis of cells identified thousands of differentially expressed genes (DEGs) between NG and SMG. DEGs down-regulated were enriched in the axonogenesis, positive regulation of cell adhesion, cell adhesion molecule and axon guidance, while SMG resulted in the up-regulation of genes were functionally associated with DNA replication, cell cycle, PI3K-Akt signaling pathway and tumorigenesis. Interestingly, some key gene terms were enriched in SMG, like hypoxia and ECM receptor interaction. Moreover, HSPC obtained from SMG culture conditions had a robust ability of proliferation in vitro. The proliferated cells also had the ability to form erythroid, granulocyte and monocyte/macrophage colonies, and can be induced to generate macrophages and megakaryocytes. In summary, our data has shown a potent impact of microgravity on hematopoietic differentiation of hPSCs for the first time and reveals an underlying mechanism for the effect of SMG on hematopoiesis development.

Background: Irisin, a novel exercise-induced myokine, was shown to mediate beneficial effects of exercise in osteoporosis. Microgravity is a major threat to bone homeostasis of astronauts during long-term spaceflight, which results in decreased bone formation. Methods: The hind-limb unloading mice model and a random position machine are respectively used to simulate microgravity in vivo and in vitro. Results: We demonstrate that not only are bone formation and osteoblast differentiation decreased, but the expression of fibronectin type III domain-containing 5 (Fdnc5; irisin precursor) is also downregulated under simulated microgravity. Moreover, a lower dose of recombinant irisin (r-irisin) (1 nM) promotes osteogenic marker gene (alkaline phosphatase (Alp), collagen type 1 alpha-1(ColIα1)) expressions, ALP activity, and calcium deposition in primary osteoblasts, with no significant effect on osteoblast proliferation. Furthermore, r-irisin could recover the decrease in osteoblast differentiation induced by simulated microgravity. We also find that r-irisin increases β-catenin expression and partly neutralizes the decrease in β-catenin expression induced by simulated microgravity. In addition, β-catenin overexpression could also in part attenuate osteoblast differentiation reduction induced by simulated microgravity. Conclusions: The present study is the first to show that r-irisin positively regulates osteoblast differentiation under simulated microgravity through increasing β-catenin expression, which may reveal a novel mechanism, and it provides a prevention strategy for bone loss and muscle atrophy induced by microgravity.

Cytoskeletal proteins and stem cell markers gene expression in human bone marrow mesenchymal stromal cells after different periods of simulated microgravity

Intestinal epithelial cell (IEC) junctions constitute a robust barrier to invasion by viruses, bacteria and exposure to ingested agents. Previous studies showed that microgravity compromises the human immune system and increases enteropathogen virulence. However, the effects of microgravity on epithelial barrier function are poorly understood. The aims of this study were to identify if simulated microgravity alters intestinal epithelial barrier function (permeability), and susceptibility to barrier-disrupting agents. IECs (HT-29.cl19a) were cultured on microcarrier beads in simulated microgravity using a rotating wall vessel (RWV) for 18 days prior to seeding on semipermeable supports to measure ion flux (transepithelial electrical resistance (TER)) and FITC-dextran (FD4) permeability over 14 days. RWV cells showed delayed apical junction localization of the tight junction proteins, occludin and ZO-1. The alcohol metabolite, acetaldehyde, significantly decreased TER and reduced junctional ZO-1 localization, while increasing FD4 permeability in RWV cells compared with static, motion and flask control cells. In conclusion, simulated microgravity induced an underlying and sustained susceptibility to epithelial barrier disruption upon removal from the microgravity environment. This has implications for gastrointestinal homeostasis of astronauts in space, as well as their capability to withstand the effects of agents that compromise intestinal epithelial barrier function following return to Earth.

We investigated the morphologic characteristics and proliferation of rabbit corneal stromal cells (CSCs) onto scaffolds under simulated microgravity. Rabbit CSCs were cultured under simulated microgravity (SMG) and static condition. Complexes of collagen-chitosan-sodium hyaluronate with pores were used as scaffolds. Rotational speed was set at 15, 20, and 30 rpm in the first, second, and third weeks of culture, respectively. Histology, immunofluorescence staining, atomic force microscope (AFM), and scanning electron microscope (SEM) examinations were performed. The cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay. In the SMG group, more CSCs adhered to the carriers in 24 hours. Confocal microscopic evaluation showed aggregated cells positively immunostained with vimentin. The SEM displayed the complex network of triangular or polygonal dendritic morphology of the cell bodies with many fine and long processes, which adhered to the scaffolds tightly. After 18 days of SMG culture, keratocyte-like CSCs with rich cell interconnections not only grew on the surface, but also into the interior of scaffolds. There were degradation phenomena in scaffolds in the SMG condition. Under static condition, cells just grew on scaffolds forming a monolayer. Cells showed elongating spindle shape and developed less processes. The absorbance values of the CCK-8 assay in the SMG group were significantly higher (P < 0.01) than in the conventional group. The condition of SMG and porous collagen-chitosan-sodium hyaluronate scaffolds facilitate the proliferation of CSCs. Cells showed robust growing characteristics and morphologic properties of keratocytes. The techniques for microgravity culture of keratocyte-like CSCs on scaffolds can yield cell aggregates or cell sheets that are favorable to the reconstruction of tissue engineering of the corneal stromal layer.

Clinostats and Random Positioning Machines (RPMs) are valuable devices for microgravity simulations in order to study fundamental gravity-dependent mechanisms on ground and in preparation for space flights. Both devices have different modes of operation, which have to be carefully considered and comprehensively discussed with respect to their potential impact on the quality of (simulated) microgravity. Here, we used the well-studied oxidative burst reaction of the immune cell (macrophage) model system, in order to compare clinorotation with random positioning. The RPM was used in a clinorotation mode, rotating the sample with 60 rpm around the horizontal axis and in the “random speed mode” (2-10 rpm) with and without random direction, thus, two axes rotating either bi- or unidirectionally. The production of Reactive Oxygen Species (ROS) during oxidative burst of the macrophages was visualized by the luminescence luminol assay. During clinorotation the cells responded with a reduction of the ROS production which is fully in line with earlier studies (clinostat, parabolic flight). In contrast, the exposure to the two random positioning modes showed that the oxidative burst response during RPM-exposure differs significantly from that observed during clinorotation, i.e. a jittering, more variant form of ROS production. We can conclude from our work, that the RPM is not suitable in its real random mode (with and without random direction; 2-10 rpm) to simulate the conditions of microgravity for the chosen system. We recommend that investigators using microgravity simulators should carefully choose the device and mode of operation specifically for their cellular system of interest.

MiR-103 inhibits osteoblast proliferation mainly through suppressing Cav1.2 expression in simulated microgravity

Exposure to microgravity has been implicated in the compromised immune function in space travellers, resulting in opportunistic infections, poor wound healing, and cancer. Since recent studies have suggested that leptin was capable of modulating immune responses, the purpose of this study was to examine effects of microgravity on the activation and proliferation of rat lung lymphocytes and then to examine the effects of leptin-mediated signal transduction mechanisms of lymphocyte activation in these same conditions. In control conditions (T-flasks cultured cells) leptin was not able by itself to increase lymphocytes proliferation, or induce significant increase of either IL-2 production or expression of lymphocytes activation markers, such as CD25 and CD71, while it markedly enhanced the positive effects induced on these parameters by concanavalin A (ConA). Using clinostatic rotating wall vessel (RWV) bioreactors to simulate a microgravity environment, we found that ConA responsiveness was inhibited. Moreover, under these conditions, leptin was not able to reverse these impaired functions. Accordingly with the above cited inhibitory effects exerted by the simulated microgravity environment, evidence was also obtained of defects in lymphocyte intracellular signal transduction induced by the incubation in RWV bioreactors, namely concerning decreased ConA-mediated PKC activity, and reduced expression of NF-kappaB, c-fos, and ERK1/2. Again, leptin appeared to be unable in restoring a physiologic increase of these parameters, different from what could be observed after complementation of the ConA-mediated signalling with phorbol myristate acetate, which instead demonstrated to overcome the inhibition of lymphocytes activating functions, in the presence of simulated microgravity conditions.

Ginsenoside Rg1 restores neural stem cell homeostasis in simulated microgravity via MAPK/AKT-vimentin axis

Regenerative life support systems (RLSS) will play a vital role in achieving self-sufficiency during long-distance space travel. Urine conversion into a liquid nitrate-based fertilizer is a key process in most RLSS. This study describes the effects of simulated microgravity (SMG) on Comamonas testosteroni, Nitrosomonas europaea, Nitrobacter winogradskyi and a tripartite culture of the three, in the context of nitrogen recovery for the Micro-Ecological Life Support System Alternative (MELiSSA). Rotary cell culture systems (RCCS) and random positioning machines (RPM) were used as SMG analogues. The transcriptional responses of the cultures were elucidated. For CO2-producing C. testosteroni and the tripartite culture, a PermaLifeTM PL-70 cell culture bag mounted on an in-house 3D-printed holder was applied to eliminate air bubble formation during SMG cultivation. Gene expression changes indicated that the fluid dynamics in SMG caused nutrient and O2 limitation. Genes involved in urea hydrolysis and nitrification were minimally affected, while denitrification-related gene expression was increased. The findings highlight potential challenges for nitrogen recovery in space.

Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity. In particular, vessel rotation speed was adjusted such that 10 mm diameter x 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall. Feasibility was demonstrated for two different cell types: cartilage and heart. Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously. Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes. Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force.